Example of Method Development

The best reference found was AOAC method 990.31 [18] that was developed by Kim and Kim. The method was based on extensive work with a variety of foods and beverages. The incentive for the work was to find an alternative to the modified Monier-WiUiams method [23], which is time-consuming and labor-intensive. 

The Kim and Kim method uses ion-exclusion chromatography with a dilute sulfuric acid eluent Detection is based on amperometric detection with a Pt working electrode. The method is quite selective. Samples are only blended with a high pH solvent to extract both bound and unbound sulfite, and then filtered and injected into the ion chromatograph. Maimitol is added to the extraction solvent to slow the oxidation of sulfite to sulfate. 

After examining the rest of the literature, several observations can be made. 

Optimum sample preparation appears to include keeping the sample tightly capped and out of sunlight until just before the injection. The sample should be quickly removed and analyzed. In general, it is better not to perform extensive sample preparation unless necessary. Of course, in many cases, sample preparation is needed. Although adding mannitol to the sample extraction solvent is not essential, it is probably important to ensure good results. 

This is just one example of how a method may be developed. There are others. 

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As an identity (ID) test, per ICH guidelines, only selectivity is required in method qualification and validation. Repeatability and intermediate precision are often included to ensure reliability of p7 determinations. Additionally, method robustness should be tested to assure that the assay performance is suitable for QC environment. Quantitative parameters such as LOD/LOQ are not required for an ID assay. If a cIEF method is used for purity determination, then all the purity parameters shown in Section 4 should be qualified. The following sections illustrate an example of method development and qualification procedures for cIEF. 

An example of method development using porous polymers is the work done for n-butyl mercaptan. n-Butyl mercaptan collected on charcoal was found to oxidize readily to the dibutyldisulfide. It was not feasible to analyze for the mercaptan as the disulfide, because the disulfide could also be present in the workplace. Silica gel was an excellent collector for the mercaptan in a dry atmosphere however, at 80% relative humidity the sorbent collected moisture preferentially, and sorbent capacity was severely reduced. 

An LC-MS/MS method [34] will be described in detail in order to serve as an example of method development and validation. This method allows the determination of some of the most common antidepressants used in clinical practice and some of their main metabolites in oral fluid and plasma samples. 

EXAMPLES OF METHODS DEVELOPMENT WITH REVERSED-PHASE SOLID-PHASE EXTRACTION... 

Examples of Methods Development with Reversed-Phase Solid-Phase Extraction... 

One example of methods developed to work around software limitations involved the requirement for a menu of cyclic nuclei as an aid in constructing structures. A partial solution to the slowness of the Graphics Tool Kit was to take advantage of the capacity for multiple screens (with only one visible at a time). 

In this section, several examples of method development procedures for protein characterization assays used for samples from biotech pharmaceutical companies are presented. 

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